细粒棘球蚴RTN4基因的克隆及序列分析

Cloning and sequence analysis of RTN4 gene of Echinococcus granulosus

【目的】克隆细粒棘球蚴(Eg)内质网膜蛋白4(Reticulon-4,RTN4)抗原基因,对其序列进行生物信息学分析。【方法】采集感染Eg的绵羊肝脏和肺脏,提取Eg头节总RNA为模板,对RTN4基因进行RT-PCR扩增,将PCR产物克隆到pMD19-T载体后测序,对其核苷酸序列及氨基酸序列进行生物信息学分析。【结果】Eg RTN4cDNA全长654bp,编码217个氨基酸,蛋白质分子质量为25.3ku,等电点为8.83。编码氨基酸序列中含有1个N端酰基化位点、2个酪蛋白激酶Ⅱ磷酸化位点和4个蛋白激酶C磷酸化位点。经生物信息学软件预测,RTN4蛋白的抗原表位区主要集中在第1-47位、第71-147位和第171-217位区域;含有2个高度疏水区,2个跨膜区,膜外区分别位于第1-47位和第171-217位。【结论】成功克隆了Eg RTN4基因,预测了其编码蛋白抗原的结构和表位。

【Objective】This study aimed to clone and carry out bioinformatics analysis on the sequence of an important antigen gene reticulon-4（RTN4）gene of Echinococcus granulosus.【Method】The total RNA was extracted by hydatid protoscolex from sheep liver and lung infected with Echinococcus granulosus.The open reading frame（ORF）sequence of RTN4 was then amplified by RT-PCR before being cloned into pMD19-T vector for bioinformation analysis of nucleotide sequence and coding sequence of amino acids.【Result】The full cDNA of RTN4 gene contained 654 bp,encoding 217 amino acids.It had a molecular weight of 25.3ku and the isoelectric point was 8.83.The coding sequence of amino acids had one N-acylation site,two casein kinaseⅡ phosphorylation sites and four protein kinase C phosphorylation sites.The results of bioinformatics software prediction showed that RTN4 protein mainly had three parts of 1to 47,71 to 147,and 171 to 217with two highly hydrophobic regions and two transmembrane domains.The extracellular domains were at 1to 47 and 171to 217.【Conclusion】The RTN4 gene of Eg was successfully cloned and the structure and antigen epitopes of its coding protein were predicted.